Wing Vein Patterning Research Articles

Wing vein patterns of the Hemiptera insectOrosanga japonicusdiffer among individuals

Although Turing's reaction-diffusion model (RD model) has been gradually accepted among biologists, application of the model is still limited. Accumulated experimental studies have shown that the morphogen gradient model can explain most patterning phenomena in embryogenesis. These experiments have been performed only in a few model animals. Therefore, it is not clear whether the discovered principle of the mechanism is generally applicable. The wing venation pattern of Drosophila melanogaster is largely determined by the morphogen gradient mechanism. We found that the gradient model cannot be applied to some other species. In the Hemiptera insect Orosanga japonicus, each individual has a unique pattern. Veins of O. japonicus extend radially from the proximal region and bifurcate to add the veins in the distal region. Interestingly, the bifurcation points are almost random and the vein number at the wing edge differs with wing size. However, the spacing between the veins is maintained evenly. Computer simulation of the RD model showed these properties do not fit the morphogen gradient model, but perfectly fit the RD model. This result suggests that the RD model may explain phenomena to which the morphogen gradient mechanism is currently believed to apply.

Mutual Inactivation of Notch Receptors and Ligands Facilitates Developmental Patterning

Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems.

Cease and desist: modulating short-range Dpp signalling in the stem-cell niche.

Drosophila ovarian germline stem cells (GSCs) are maintained by the extracellular BMP2/4 orthologue Dpp, which is produced from the surrounding somatic niche. The Dpp signal has a short range; it induces a response in GSCs within the niche, but is rapidly extinguished in their progeny only one cell-diameter away. To ensure the correct balance between stem-cell maintenance and differentiation, several regulatory mechanisms that modulate the Dpp signal at many stages of the pathway have been described. Here, we discuss the nature of the ovarian Dpp signal and review the catalogue of mechanisms that regulate it, demonstrating how the exquisite modulation of Dpp signalling in this context can result in precise and robust control of stem-cell fate. This modulation is applicable to other stem-cell environments that use BMPs as a niche signal, and the regulatory mechanisms are conceptually relevant to several other stem-cell systems.

A Genome-Wide Screen Reveals a Role for microRNA-1 in Modulating Cardiac Cell Polarity

Many molecular pathways involved in heart disease have their roots in evolutionarily ancient developmental programs that depend critically on gene dosage and timing. MicroRNAs (miRNAs) modulate gene dosage posttranscriptionally, and among these, the muscle-specific miR-1 is particularly important for developing and maintaining somatic/skeletal and cardiac muscle. To identify pathways regulated by miR-1, we performed a forward genetic screen in Drosophila using wing-vein patterning as a biological assay. We identified several unexpected genes that genetically interacted with dmiR-1, one of which was kayak, encodes a developmentally regulated transcription factor. Additional studies directed at this genetic relationship revealed a previously unappreciated function of dmiR-1 in regulating the polarity of cardiac progenitor cells. The mammalian ortholog of kayak, c-Fos, was dysregulated in hearts of gain- or loss-of-function miR-1 mutant mice in a stress-dependent manner. These findings illustrate the power of Drosophila-based screens to find points of intersection between miRNAs and conserved pathways in mammals.

Feedback regulation ofDrosophilaBMP signaling by the novel extracellular protein Larval Translucida

The cellular response to the Drosophila BMP 2/4-like ligand Decapentaplegic (DPP) serves as one of the best-studied models for understanding the long-range control of tissue growth and pattern formation during animal development. Nevertheless, fundamental questions remain unanswered regarding extracellular regulation of the ligand itself, as well as the nature of the downstream transcriptional response to BMP pathway activation. Here, we report the identification of larval translucida (ltl), a novel target of BMP activity in Drosophila. Both gain- and loss-of-function analyses implicate LTL, a leucine-rich repeat protein, in the regulation of wing growth and vein patterning. At the molecular level, we demonstrate that LTL is a secreted protein that antagonizes BMP-dependent MAD phosphorylation, indicating that it regulates DPP/BMP signaling at or above the level of ligand-receptor interactions. Furthermore, based on genetic interactions with the DPP-binding protein Crossveinless 2 and biochemical interactions with the glypican Dally-like, we propose that LTL acts in the extracellular space where it completes a novel auto-regulatory loop that modulates BMP activity.

P31. Directional transport of Dpp/BMP draws diversified wing vein patterns in insects

A fundamental question in biology is to uncover the mechanisms underlying how conserved signalling molecules generate diversified morphologies. It has been shown that a conserved Bone Morphogenetic Protein (BMP) type-ligand Decapentalegic (Dpp) functions as a wing vein determinant in the fruit fly, however it remains unknown how “diffusible” Dpp specifies elaborate and diversified insect wing vein patterns. Here we show that tightly controlled extracellular distribution of Dpp, rather than its expression pattern, correlates with complex wing vein patterns in two distantly related species, the Dipteran Drosophila melanogaster (fruit fly) and the Hymenopteran Athalia rosae (sawfly). In Drosophila , directional diffusion and active retention of Dpp localize the signal in future wing veins through BMP binding proteins, Short gastrulation (Sog) and Crossveinless (Cv), and through BMP type I receptor Thickvein (Tkv) and unknown positive feedback mechanisms, respectively. In sawfly, Dpp signal also reflects more complex wing vein patterns and loss of Cv by RNAi fails to establish both distinct fore- and hind-wing vein patterns. These results suggest that diversified insect wing vein patterns are acquired through directional Dpp diffusion at the extracellular space. Changes in extracellular regulation of secreted growth factors may be a general mechanism for creating evolutionarily diversified patterns.

Rabex-5 Ubiquitin Ligase Activity Restricts Ras Signaling to Establish Pathway Homeostasis in Drosophila

The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders [1, 2], and in mature tissues, it can lead to cancer [3-5]. We identify Rabex-5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. We show that Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking [6] and ubiquitin ligase activity [7, 8]. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis.

03-P051 Facilitated transport and active retention of BMP draw diversified wing vein patterns in insects

03-P051 Facilitated transport and active retention of BMP draw diversified wing vein patterns in insects

Multiple Allelism of the net Gene in Drosophila melanogaster and Drosophila simulans

The net gene mutations are known to cause abnormal pattern of veining in all wing regions except for the first posterior cells. In natural populations of Drosophila melanogaster, the net alleles were identified, which differ in phenotypic expression from standard mutations. The mutants net-extra-analis from a population Belokurikha-2000 have only a single additional vein in the third posterior cell. A line from Chernobyl-1986 population have another nontypical allele netCh86 and shows a lower degree of abnormalities than that usually observed. About 10% of these flies have an additional vein fragment in the first posterior cell. In both males and females of D. simulans population Tashkent-2001, which exhibit netST91 mutation, a net of additional veins is formed as a specific additional fragment in the first posterior cell. The pattern of veining conferred by alleles net-extra-analis and netCh86 is altered to a lesser extent; these alleles are dominant with respect to alleles net2-45 and netST91, which cause more abnormalities. The heterozygotes for alleles netST9 and netCh86 and for Df(2) net62 deletion have an additional fragment in the first posterior cell and show similarly strong deviations from normal wing vein pattern. The natural net alleles correspond, presumably, to different molecular gene defects involved into uncertain local interactions with numerous modifying factors and other genes that specify the wing vein pattern.

The wings of Bombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report.

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm, Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development in B. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike in Drosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adult Drosophila wings. Partially excised wing discs did not show in situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early in B. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades in B. mori unlike evagination of only the pouch region as wing blades seen in Drosophila.

The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development.

Drosophila nemo was first identified as a gene required for tissue polarity during ommatidial development. We have extended the analysis of nemo and found that it participates in multiple developmental processes. It is required during wing development for wing shape and vein patterning. We observe genetic interactions between nemo and mutations in the Notch, Wingless, Frizzled and Decapentaplegic pathways. Our data support the findings from other organisms that Nemo proteins act as negative regulators of Wingless signaling. nemo mutations cause polarity defects in the adult wing and overexpression of nemo leads to abdominal polarity defects. The expression of nemo during embryogenesis is dynamic and dsRNA inhibition and ectopic expression studies indicate that nemo is essential during embryogenesis.

Regulation of Drosophila wing vein patterning: net encodes a bHLH protein repressing rhomboid and is repressed by rhomboid-dependent Egfr signalling.

The stereotyped pattern of veins in the Drosophila wing is generated in response to local EGF signalling. Mutations in the rhomboid (rho) gene, which encodes a sevenpass membrane protein required to enhance signalling transmitted by the EGF receptor (Egfr), inhibit vein development and disrupt the vein pattern. By contrast, net mutations produce ectopic veins in intervein regions. We have cloned the net gene and show that it encodes a basic HLH protein that probably acts as a transcriptional repressor. net and rho are expressed in mutually exclusive patterns during the development of the wing imaginal disc. Lack of net activity causes rho expression to expand, and vice versa. Furthermore, ectopic expression of net or rho results in their mutual repression and thus suppresses vein formation or generates tube-like wings composed of vein-like tissue. Egfr signalling and net exert mutually antagonising activities during the specification of vein versus intervein fate. While Egfr signalling represses net transcription, net exhibits a two-tiered control by repressing rho transcription and interfering with Egfr signalling downstream of Rho. Our results further suggest that net is required to maintain intervein development by restricting Egfr signalling, which promotes vein development, to the Net-free vein regions of the wing disc.

Restricted patterning of vestigial expression in Drosophila wing imaginal discs requires synergistic activation by both Mad and the drifter POU domain transcription factor.

The Drosophila Vestigial protein has been shown to play an essential role in the regulation of cell proliferation and differentiation within the developing wing imaginal disc. Cell-specific expression of vg is controlled by two separate transcriptional enhancers. The boundary enhancer controls expression in cells near the dorsoventral (DV) boundary and is regulated by the Notch signal transduction pathway, while the quadrant enhancer responds to the Decapentaplegic and Wingless morphogen gradients emanating from cells near the anteroposterior (AP) and DV boundaries, respectively. MAD-dependent activation of the vestigial quadrant enhancer results in broad expression throughout the wing pouch but is excluded from cells near the DV boundary. This has previously been thought to be due to direct repression by a signal from the DV boundary; however, we show that this exclusion of quadrant enhancer-dependent expression from the DV boundary is due to the absence of an additional essential activator in those cells. The Drosophila POU domain transcriptional regulator, Drifter, is expressed in all cells within the wing pouch expressing a vgQ-lacZ transgene and is also excluded from the DV boundary. Viable drifter hypomorphic mutations cause defects in cell proliferation and wing vein patterning correlated with decreased quadrant enhancer-dependent expression. Drifter misexpression at the DV boundary using the GAL4/UAS system causes ectopic outgrowths at the distal wing tip due to induction of aberrant Vestigial expression, while a dominant-negative Drifter isoform represses expression of vgQ-lacZ and causes severe notching of the adult wing. In addition, we have identified an essential evolutionarily conserved sequence element bound by the Drifter protein with high affinity and located adjacent to the MAD binding site within the quadrant enhancer. Our results demonstrate that Drifter functions along with MAD as a direct activator of Vestigial expression in the wing pouch.

The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development.

TGF-beta-related signaling pathways play diverse roles during vertebrate and invertebrate development. A common mechanism for regulating the activity of TGF-beta family members is inhibition by extracellular antagonists. Recently, the Drosophila short gastrulation (sog) gene was shown to encode a predicted diffusible factor which antagonizes signaling mediated by the TGF-beta-like Decapentaplegic (Dpp) pathway in the early blastoderm embryo. sog and dpp, which are among the earliest zygotic genes to be activated, are expressed in complementary dorsal-ventral domains. The opposing actions of sog and dpp in the early embryo have been highly conserved during evolution as their vertebrate counterparts, chordin and BMP-4, function homologously to define neural versus non-neural ectoderm in Xenopus. Here we exploit the genetically sensitive adult wing vein pattern to investigate the generality of the antagonistic relationship between sog and dpp. We show that dpp is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating Dpp feedback loop. These data support the view that Sog is a dedicated Dpp antagonist.

Patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets.

The membrane protein, Patched, plays a critical role in patterning embryonic and imaginal tissues in Drosophila. patched constitutively inactivates the transcription of target genes such as wingless, decapentaplegic, and patched itself. The secreted protein, Hedgehog, induces transcription of target genes by opposing the Patched signaling pathway. Using the Gal4 UAS system we have overexpressed patched in wing imaginal discs and found that high Patched levels, expressed in either normal or ectopic patterns, result in loss of wing vein patterning in both compartments centering at the anterior/posterior border. In addition, patched inhibits the formation of the mechanosensory neurons, the campaniform sensilla, in the wing blade. The patched wing vein phenotype is modulated by mutations in hedgehog and cubitus interruptus (ci). Patched overexpression inhibits transcription of patched and decapentaplegic and post-transcriptionally decreases the amount of Ci protein at the anterior/posterior boundary. In hedgehogMrt wing discs, which express ectopic hedgehog, Ci levels are correspondingly elevated, suggesting that hedgehog relieves patched repression of Ci accumulation. Protein kinase A also regulates Ci; protein kinase A mutant clones in the anterior compartment have increased levels of Ci protein. Thus patched influences wing disc patterning by decreasing Ci protein levels and inactivating hedgehog target genes in the anterior compartment.

Roles of the Notch gene in Drosophila wing morphogenesis

The Notch gene encodes a transmembrane protein that functions as a receptor of intercellular signals in many developmental processes of Drosophila. We study here the Notch function in wing morphogenesis and vein patterning in genetic mosaics of both Notch null and Notch gain-of-function alleles. Cell proliferation and differentiation properties of mutant Notch cells define three different Notch requirements in the wing: in imaginal disc cell proliferation, in restriction of vein differentiation and in margin formation. The study of Notch mosaics in different mutant backgrounds reveals that Notch activity during epidermal cell proliferation and wing vein differentiation is exerted by its regulation of a common group of genes involved in the specification and restriction of vein competent regions.

Second-site modifiers of the Delta wing phenotype in Drosophila melanogaster.

We have screened for dominant enhancers and suppressors of the wing phenotype associated with two Delta alleles: Dl 9P39, an amorphic allele, and Dl FE32, an antimorphic allele. The interactions of some of the modifiers with Delta are due to haplo-insufficient expression of the corresponding genes. Although not explicitly shown for the remaining cases, we assume that haploin-sufficiency is also the basis for the relationships of these genes to Delta, since no allele specific interactions were observed. The modifiers found define 22 genes with pleiotropic expression, which can be classified into two groups: genes required for wing vein pattern formation and for neurogenesis, and genes which are not required for neurogenesis. Among the genes of the first group, Hairless and Star were previously known to participate in neural development. One further modifier was found which may correspond to a new neurogenic gene. The second group of genes is larger and includes already known loci, e.g., Plexate, blistered, plexus, etc, as well as other previously unidentified genes, which function during wing morphogenesis.

Genetic analysis of the wing vein pattern of Drosophila.

Of the many mutations known to affect the wing vein pattern we have selected the most extreme in 29 genes for study. Their phenotype can be classified in two major classes: lack-of-veins and excess-of-veins, and in several internally coherent groups. The study of multiple mutant combinations, within groups and between groups, reveals several genetic operations at work in the generation of the vein pattern. The finding that some of these mutations also affect cell proliferation in characteristic ways has prompted a generative model of wing morphogenetic and pattern formation based on cell behaviour properties defined by the corresponding wild-type genes.

Pattern compensation in Drosophila wing vein development

Much interest in developmental genetics has focused on the formation of patterns. Using the wing vein pattern of Drosophila melanogasler as our model system, we show that vein placement is essentially independent of cell lineage. In studying vein placement in a variety of wing vein mutants, we observed that the position of a vein can move to compensate for the loss of vein in nearby parts of the wing. These observations led us to consider an interacting diffusion gradient model of vein formation.